Monday, March 19, 2012

Dynamics and Sloppiness in Protein Synthesis

First, there's the initiation stage when a ribosome binds to the initiation codon in messenger RNA (mRNA) and the translation initiation complex forms by recruiting additional components.

Second, there's the elongation stage when the elongation complex moves along the mRNA translating the coding region and producing a polypeptide chain. The elongation rate is relatively constant but from time to time the elongation complex pauses at particular sites that are difficult to translate.

Finally, the elongation complex encounters a termination site where it disassembles and the ribosome with its various factors is released from the mRNA.

At any given time, a typical mRNA molecule will contain ribosomes and translation factors at all three stages. If initiation, elongation, and termination are rapid then each mRNA will be translated many times before it is degraded and there will be several elongation complexes working simultaneously. This gives rise to a "polysome" as shown in the figure below where multiple mRNAs are being translated in E. coli.

I the past we've had some estimates of the frequency of initiation by studying some specific mRNAs. We also know something about the rate of elongation from work on translation in vitro.

For many years it has been possible to treat cells with drugs that block elongation, for example, then isolate polysomes and digest all exposed RNA with nucleases. You are left with elongation complexes and small bits of mRNA that have been protected from the nuclease because they are bound to the complex. These little bits of RNA can be copied into DNA, cloned, and matched to the sequence of a particular gene. The result is a ribosome profile showing the average position of a translation complex inside the cell. One can also block initiation and ask how many ribosome are sitting at an initiation site at any given time. Same for termination.

Modern sequencing technology ("deep sequencing") allows for this type of analysis at a global level. Millions of protected RNAs are sequenced and matched to the genome of the species. This way you get information on ribosome profiling for thousands of genes at the same time.

Ingolia et al. (2011) did this experiment using mouse cells. There's a brief summary in Science (Weiss and Atkins, 2011).

Translation Rate

The workers blocked translation initiation with a drug called harringtonine then waited for various lengths of time before stopping elongation with cycloheximide. As the elongation complexes moved down the mRNA they left longer and longer exposed regions at the 5′ end of the molecule. An analysis of several thousand genes yielded an average rate of elongation of 5.6 codons per second or about 17 nucleotides per second.

This rate is a lot slower than the rate in bacteria. In E. coli, for example, the rate of protein synthesis is about 18 amino acid residues per second, which corresponds to a rate of movement of 54 nucleotides per second. This is the same as the rate of transcription (55 nucleotides per second) and that's why protein synthesis can keep up with transcription (see figure).

Pause Sites

About 25% of all genes have, on average, one strong pause site where translation complexes halt for several seconds. Most pause sites have a glutamate or aspartate codon at the A site and a proline or glycine codon immediately downstream (already translated). An additional proline codon usually lies two codons downstream.

We used to think that pause sites were caused by rare codons where the abundance of the corresponding aminoacyl-tRNA was much lower than normal but apparently that's not correct.

There's no evidence of pileup at a pause site nor is there evidence of a depleted region downstream of a pause site ("shadow"). Pileup and depletion would be expected if translating ribosome were densely packed on an mRNA molecule as they are in bacteria. The evidence suggests that the average ribosome density on mRNA is not high enough to permit a trailing ribosome to catch up with one that's paused for several seconds. This, in turn, indicates that translation initiation is much slower in the mouse cells than in bacteria.

Initiation Sies

The most starling result of this study is the number of unannotated translation initiation sites. Recall that most start sites have the methionine codon AUG and in eukaryotes the pre-initiation complex binds to the 5′ cap and then moves down the mRNA until it encounters the first AUG where it pauses while the complete initiation complex is assembled. Then translation begins at this site.

Ingolia et al. discovered that 65% of their mRNAs (genes) contain more than one site where ribosomes are bound in the presence of the drug harringtonine—a drug that prevents translation initiation complexes from transitioning to elongation complexes. They refer to these as initiation sites but I'm not sure that's valid unless they have additional evidence that translation actually begins at these sites. Many of the sites cover codons that differ from AUG by a single nucleotide.

Most of these sites are downstream of the annotated start site, suggesting that many proteins with truncated N-terminii are being produced. However, in 280 genes there is no detectable binding at the annotated start site indicating that a downstream site is preferred or that the annotation (and/or sequence) is incorrect.

That's a problem with these studies. When the results conflict with the annotation you never know for sure whether there's a genuine conflict or whether the annotation is wrong. Most of these genes have not been well studied and there could be several mistakes: (a) the sequence could be wrong, (b) the transcription start site could be wrong, or (c) the predicted spliced products could be wrong.

Some of these issues could be resolved by looking at each individual gene but that's a lot of work when dealing with results from 5000 genes. Hopefully, there will be more studies like this on other types of mouse cells and on other species.

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Laurence A. Moran

Larry Moran is a Professor in the Department of Biochemistry at the University of Toronto. You can contact him by looking up his email address on the University of Toronto website.

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Quotations

The old argument of design in nature, as given by Paley, which formerly seemed to me to be so conclusive, fails, now that the law of natural selection has been discovered. We can no longer argue that, for instance, the beautiful hinge of a bivalve shell must have been made by an intelligent being, like the hinge of a door by man. There seems to be no more design in the variability of organic beings and in the action of natural selection, than in the course which the wind blows.Charles Darwin (c1880)Although I am fully convinced of the truth of the views given in this volume, I by no means expect to convince experienced naturalists whose minds are stocked with a multitude of facts all viewed, during a long course of years, from a point of view directly opposite to mine. It is so easy to hide our ignorance under such expressions as "plan of creation," "unity of design," etc., and to think that we give an explanation when we only restate a fact. Any one whose disposition leads him to attach more weight to unexplained difficulties than to the explanation of a certain number of facts will certainly reject the theory.

Charles Darwin (1859)Science reveals where religion conceals. Where religion purports to explain, it actually resorts to tautology. To assert that "God did it" is no more than an admission of ignorance dressed deceitfully as an explanation...

Quotations

The world is not inhabited exclusively by fools, and when a subject arouses intense interest, as this one has, something other than semantics is usually at stake.
Stephen Jay Gould (1982)
I have championed contingency, and will continue to do so, because its large realm and legitimate claims have been so poorly attended by evolutionary scientists who cannot discern the beat of this different drummer while their brains and ears remain tuned to only the sounds of general theory.
Stephen Jay Gould (2002) p.1339
The essence of Darwinism lies in its claim that natural selection creates the fit. Variation is ubiquitous and random in direction. It supplies raw material only. Natural selection directs the course of evolutionary change.
Stephen Jay Gould (1977)
Rudyard Kipling asked how the leopard got its spots, the rhino its wrinkled skin. He called his answers "just-so stories." When evolutionists try to explain form and behavior, they also tell just-so stories—and the agent is natural selection. Virtuosity in invention replaces testability as the criterion for acceptance.
Stephen Jay Gould (1980)
Since 'change of gene frequencies in populations' is the 'official' definition of evolution, randomness has transgressed Darwin's border and asserted itself as an agent of evolutionary change.
Stephen Jay Gould (1983) p.335
The first commandment for all versions of NOMA might be summarized by stating: "Thou shalt not mix the magisteria by claiming that God directly ordains important events in the history of nature by special interference knowable only through revelation and not accessible to science." In common parlance, we refer to such special interference as "miracle"—operationally defined as a unique and temporary suspension of natural law to reorder the facts of nature by divine fiat.
Stephen Jay Gould (1999) p.84

Quotations

My own view is that conclusions about the evolution of human behavior should be based on research at least as rigorous as that used in studying nonhuman animals. And if you read the animal behavior journals, you'll see that this requirement sets the bar pretty high, so that many assertions about evolutionary psychology sink without a trace.

Jerry Coyne
Why Evolution Is TrueI once made the remark that two things disappeared in 1990: one was communism, the other was biochemistry and that only one of them should be allowed to come back.

Sydney Brenner
TIBS Dec. 2000
It is naïve to think that if a species' environment changes the species must adapt or else become extinct.... Just as a changed environment need not set in motion selection for new adaptations, new adaptations may evolve in an unchanging environment if new mutations arise that are superior to any pre-existing variations

Douglas Futuyma
One of the most frightening things in the Western world, and in this country in particular, is the number of people who believe in things that are scientifically false. If someone tells me that the earth is less than 10,000 years old, in my opinion he should see a psychiatrist.

Francis Crick
There will be no difficulty in computers being adapted to biology. There will be luddites. But they will be buried.

Sydney Brenner
An atheist before Darwin could have said, following Hume: 'I have no explanation for complex biological design. All I know is that God isn't a good explanation, so we must wait and hope that somebody comes up with a better one.' I can't help feeling that such a position, though logically sound, would have left one feeling pretty unsatisfied, and that although atheism might have been logically tenable before Darwin, Darwin made it possible to be an intellectually fulfilled atheist

Richard Dawkins
Another curious aspect of the theory of evolution is that everybody thinks he understand it. I mean philosophers, social scientists, and so on. While in fact very few people understand it, actually as it stands, even as it stood when Darwin expressed it, and even less as we now may be able to understand it in biology.

Jacques Monod
The false view of evolution as a process of global optimizing has been applied literally by engineers who, taken in by a mistaken metaphor, have attempted to find globally optimal solutions to design problems by writing programs that model evolution by natural selection.